Chloramphenicol degrading strain CS1 and application thereof in repairing environment polluted by chloramphenicol, thiamphenicol and nitrobenzene compound

Abstract

The invention discloses a chloramphenicol degrading strain CS1 and application thereof in repairing the environment polluted by chloramphenicol, thiamphenicol and nitrobenzene compounds, the chloramphenicol degrading strain CS1 is preserved in China Center for Type Culture Collection on March 10, 2025, the preservation number is CCTCCM2025425, and the preservation address is Luojia mountain on eight road of Wuchang District, Wuhan City, Hubei Province. The chloramphenicol degrading strain CS1 disclosed by the invention can be used for completely removing the chloramphenicol which is up to 100 mg / L in an inorganic salt culture medium within 150 minutes. The strain keeps efficient chloramphenicol degradation activity in a water body at the temperature of 15-50 DEG C and the pH value of 4-11. Meanwhile, the strain can degrade chloramphenicol in soil within a short time, and long-acting chloramphenicol degradation activity is kept. The invention can be used for solving the problem of harm of residual chloramphenicol to soil environment, crops and human health.

Description

Technical Field

[0001] The present invention belongs to the technical field of biodegradation, and particularly relates to a chloramphenicol-degrading strain CS1 and its application in repairing environments polluted by chloramphenicol, thiamphenicol, and nitrobenzene compounds. Background Art

[0002] Antibiotics have played an important role in combating infectious diseases, but have also led to the spread of antimicrobial resistance. As the first large-scale produced antibiotic, chloramphenicol has been widely used in human and veterinary clinical fields. Its extensive use has ultimately led to a chloramphenicol pollution level as high as 4.8 mg / kg in farmland soil, posing a serious threat to ecological safety and human health. It is urgent to eliminate chloramphenicol pollution from polluted soil.

[0003] Microbial degradation is an economical and eco-friendly method for removing soil pollutants. So far, resistant bacteria capable of transforming chloramphenicol have been discovered, but the accumulated metabolites still pose a threat to human health. Currently, several strains reported to be able to mineralize chloramphenicol mainly originate from activated sludge, and their application prospects in polluted soil sites are not yet clear. In addition, the complete mineralization pathway of chloramphenicol has not been elucidated.

[0004] The environmental factors in actual polluted sites pose significant challenges to the effectiveness of bioaugmentation, among which temperature and pH are key limiting factors affecting the remediation rate. Therefore, screening for highly efficient chloramphenicol-degrading strains and studying their degradation characteristics and environmental tolerance are of great significance for the treatment and remediation of chloramphenicol pollution. Summary of the Invention

[0005] The purpose of this part is to outline some aspects of the embodiments of the present invention and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this part, as well as in the abstract and title of the present application, to avoid obscuring the purpose of this part, the abstract, and the title, but such simplifications or omissions cannot be used to limit the scope of the present invention.

[0006] In view of the above and / or problems existing in the prior art, the present invention is proposed.

[0007] Therefore, the purpose of the present invention is to overcome the deficiencies in the prior art and provide a Nocardia testacea CS1, which was deposited at the China Center for Type Culture Collection on March 10, 2025, with the deposit number CCTCC M 2025425 and the deposit address being Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0008] Another purpose of the present invention is to overcome the deficiencies in the prior art and provide a degrading bacterium agent, characterized in that it contains the above-mentioned Nocardia testacea.

[0009] Another object of the present invention is to overcome the deficiencies in the prior art and provide an application of Nocardia fulva or the bacterial agent in repairing the environment polluted by chloramphenicol, thiamphenicol and nitrobenzene compounds.

[0010] As a preferred embodiment of the preparation method of the present invention, wherein: the Nocardia fulva or the bacterial agent described in claim 2 is added to an environment containing chloramphenicol, thiamphenicol and nitrobenzene compounds for reaction.

[0011] As a preferred embodiment of the preparation method of the present invention, wherein: the pH of the reaction is 4 to 11.

[0012] As a preferred embodiment of the preparation method of the present invention, wherein: the reaction temperature is 30 °C.

[0013] As a preferred embodiment of the preparation method of the present invention, wherein: the pH of the reaction is 7 to 8.

[0014] As a preferred embodiment of the preparation method of the present invention, wherein: the concentration of chloramphenicol in the environment is 100 mg / L.

[0015] Another object of the present invention is to overcome the deficiencies in the prior art and provide an application of Nocardia fulva in preparing a product for removing chloramphenicol, thiamphenicol and nitrobenzene compounds from water and soil.

[0016] Advantages of the present invention:

[0017] The present invention has isolated and screened a chloramphenicol-degrading strain CS1, which can completely remove up to 100 mg / L of chloramphenicol in an inorganic salt medium within 150 minutes. This strain maintains high-efficiency chloramphenicol-degrading activity in the range of 15 - 50 °C and pH 4 - 11 in water. At the same time, this strain can degrade chloramphenicol in soil in a short time and maintain long-term chloramphenicol-degrading activity. The present invention can be used to solve the problems of the harm of residual chloramphenicol to the soil environment, crops and human health. Description of the drawings

[0018] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings. Among them:

[0019] Figure 1 It is a photograph of the enrichment solution and the biofilm therein of the embodiment of the present invention, wherein the red circle is the biofilm.

[0020] Figure 2For the dynamic change of chloramphenicol concentration when the strain CS1 in the embodiment of the present invention degrades 100 mg / L chloramphenicol.

[0021] Figure 3 For the photo of the strain CS1 in the embodiment of the present invention cultured on an LB solid plate at 30 °C for 7 days.

[0022] Figure 4 For the scanning electron microscope photo of the strain CS1 in the embodiment of the present invention.

[0023] Figure 5 For the granular bacteria of the strain CS1 in the embodiment of the present invention in an LB broth medium.

[0024] Figure 6 For the liquid chromatography tandem mass spectrometry (LC-MS / MS) spectrum of the metabolites of the strain CS1 in the embodiment of the present invention when degrading chloramphenicol.

[0025] Figure 7 For the metabolic pathway of the strain CS1 in the embodiment of the present invention when degrading chloramphenicol.

[0026] Figure 8 For the relative concentration change of the degradation of 20 mg / L chloramphenicol by 21 mg of the strain CS1 in 50 mL of an inorganic salt medium under different temperature conditions in the embodiment of the present invention.

[0027] Figure 9 For the relative concentration change of the degradation of 20 mg / L chloramphenicol by 10 mg of the strain CS1 in 50 mL of an inorganic salt medium under different pH conditions in the embodiment of the present invention.

[0028] Figure 10 For the determination of the mineralization effect of the pulse pollution of 14C-labeled chloramphenicol in paddy soil cultured at 15 °C by the strain CS1 in the embodiment of the present invention.

[0029] Figure 11 For the determination of the mineralization effect of the pulse pollution of 14C-labeled chloramphenicol in red soil cultured at 30 °C by the strain CS1 in the embodiment of the present invention.

[0030] Figure 12 For the analysis of the degradation performance of the strain CS1 in the embodiment of the present invention on common chloramphenicol and p-nitrophenyl organic pollutants. Detailed implementation manners

[0031] To make the above objects, features, and advantages of the present invention more obvious and understandable, the following detailed description of the specific implementation manners of the present invention is provided in conjunction with the embodiments of the specification.

[0032] In the following description, numerous specific details are set forth to provide a thorough understanding of the present invention. However, the present invention may be practiced in other ways different from those described herein. Persons skilled in the art can make similar extensions without departing from the spirit of the present invention. Therefore, the present invention is not limited by the specific embodiments disclosed below.

[0033] Secondly, the so-called "one embodiment" or "embodiment" herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation manner of the present invention. The appearances of "in one embodiment" in different places in this specification do not all refer to the same embodiment, nor are they separate or alternative embodiments that exclude each other with other embodiments.

[0034] Unless otherwise specified, the raw materials used in the embodiments of the present invention are all commercially available. Details are shown in Table 1.

[0035] Table 1

[0036]

[0037]

[0038] High - performance liquid chromatography verification method for chloramphenicol degradation effect: Add an equal volume of methanol to the culture solution and filter it with a filter membrane (pore size 0.22 μm). Use a Waters e2695 high - performance liquid chromatograph to measure the content of chloramphenicol in the filtrate. Liquid chromatography conditions: The volume ratio of mobile phase A (chromatographic acetonitrile) and mobile phase B (0.1% chromatographic formic acid aqueous solution, V:V) is 35:65. A Symmetry C18 reverse - phase column (5 μm, 4.6 mm × 250 mm, Waters Co., USA), column temperature is 40 °C, a Waters e2487 ultraviolet - visible light detector, detection wavelength 270 nm, injection volume 10 μL, and flow rate 1.0 mL / min. Quantify by peak area using the external standard method.

[0039] The formula of the inorganic salt medium (1 L) is: 2.44 g / L Na2HPO4, 1.52 g / L KH2PO4, 0.5 g / L (NH4)2SO4, 0.2 g / L MgSO4, 0.05 g / L CaCl2·2H2O, 5.0 mg / L EDTA, 2.0 mg / L FeSO4·7H2O, 0.9 mg / L ZnSO4·7H2O, 0.27 mg / L MnCl2·4H2O, 2.7 mg / L H3BO3, 1.8 mg / L CoCl2·6H2O, 0.09 mg / L CuCl2·2H2O, 0.18 mg / L NiCl2·6H2O, and 0.27 mg / L Na2MoO4·2H2O are dissolved in 1 L of distilled water, pH 7.0.

[0040] The LB solid agar formulation is as follows: 10 g of NaCl, 5 g of yeast extract, 10 g of tryptone, 18 g of agar powder, add distilled water to 1 L, pH 7.0.

[0041] The LB broth medium (1 L) formulation is as follows: 10 g of NaCl, 5 g of yeast extract, 10 g of tryptone, add deionized water to 1 L, pH 7.0.

[0042] The present invention provides a Nocardia testacea CS1, which was deposited at the China Center for Type Culture Collection on March 10, 2025, with the deposit number CCTCC M 2025425 and the deposit address being Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0043] Example 1

[0044] Isolation and identification of the chloramphenicol-degrading strain CS1:

[0045] Collect soil samples from farmland in Changsha, Hunan, air-dry, grind and sieve (<0.85 mm). Weigh 2.0 g of soil and add it to 20 mL of inorganic salt medium, stir evenly to make an inoculum. Add 2 mL of the inoculum to 20 mL of inorganic salt medium containing 20 mg / L of chloramphenicol, culture at 30 °C and 150 rpm for 20 days, transfer to fresh sterile inorganic salt medium at an inoculum amount of 10% (V / V), and transfer continuously for five times. Pick the white biofilm growing on the surface of the fifth-generation enrichment solution, as Figure 1 shown, break it and spread it on an LB agar plate, culture at 30 °C for 20 days, pick single colonies on the plate and inoculate them into inorganic salt medium containing 20 mg / L of chloramphenicol, culture at 30 °C and 150 rpm for 20 days, and then use a high-performance liquid chromatography (HPLC) to detect whether each single colony has the function of degrading chloramphenicol. Through verification, 1 chloramphenicol-degrading strain was obtained and named CS1. The degradation rate of this strain to 100 mg / L of chloramphenicol reached 100% in 2.5 hours ( Figure 2 ). The colony morphology of strain CS1 on the LB solid plate is ( Figure 3 ): white, round, the colony surface is rough, and slightly convex in the middle. The scanning electron microscope photo of strain CS1 is as Figure 4 shown, presenting as short rod-shaped. The whole-genome sequence of strain CS1 was obtained through second-generation sequencing and third-generation sequencing, and the average nucleotide identity with Nocardia testacea NBRC100365T was as high as 96.1%. Strain CS1 was named Nocardia testacea CS1.

[0046] Example 2

[0047] Preparation method of the degrading agent of strain CS1:

[0048] The degradation strain CS1 isolated and screened in Example 1 was streaked on an LB agar plate. After culturing for 7 days, the CS1 colony biomass was inoculated into 100 mL of LB broth medium and cultured with shaking at 30 °C and 150 rpm for 7 days. As Figure 5 shown, granular bacteria were produced.

[0049] Shaking was stopped, and the bacteria were allowed to settle naturally to the bottom. The upper liquid was removed, and the degradation bacterial agent was obtained.

[0050] Example 3

[0051] Determination of the chloramphenicol mineralization pathway of strain CS1:

[0052] The metabolites in the chloramphenicol degradation solution in Example 1 were analyzed and identified using high-performance liquid chromatography (Infinity 1260, Agilent, USA) tandem quadrupole time-of-flight mass spectrometry (TripleTOF 5600, AB Sciex LLC, USA) (HPLC–QTOF-MS). The HPLC–QTOF-MS conditions were as follows: the chromatographic column was an X-Bridge C18 column (2.1 mm × 100 mm; 3.5 μm), the volume ratio of mobile phase A (chromatographic methanol) and mobile phase B (0.1% formic acid aqueous solution, V:V) was 20:80, the column temperature was 20 °C, the injection volume was 1 μL, and the flow rate was 0.2 mL / min. The ion source was ESI–, and the scanning range was 30–500 m / z.

[0053] As Figures 6 - 7 shown, strain CS1 degraded 100 mg / L chloramphenicol to produce 14 metabolites such as 2,2-dichloro-N-[1-hydroxy-1-(4-nitrophenyl)-3-oxo-2-propyl]acetamide (TP-319), 2-(2,2-dichloroacetamido)-3-hydroxy-3-(4-nitrophenyl)propionic acid (TP-335), 2-amino-3-hydroxy-3-(4-nitrophenyl)propionic acid (TP-225), 4-nitrobenzaldehyde (TP-151), 4-nitrobenzoic acid (TP-166), 4-nitroso-benzoic acid (TP-150), 4-(hydroxyamino)benzoic acid (TP-152), 4-aminobenzoic acid (TP-136), protocatechuic acid (TP-153), 2,2-dichloro-N-(2-hydroxyethyl)acetamide (TP-170), 2,2-dichloro-N-(2-oxoethyl)acetamide (TP-168), N-(dichloroacetyl)glycine (TP-184), and dichloroacetic acid (TP-127). Strain CS1 initiated the degradation of chloramphenicol through two pathways, namely, hydroxylation at the C3 position and cleavage of the C1-C2 bond ( Figure 7) It is speculated that both pathways generate TP-151, TP-127 and glycine. Among them, TP-127 and glycine are mineralized, while TP-151 is finally mineralized through the protocatechuic acid metabolic pathway after nitro reduction and deamination.

[0054] Example 4

[0055] Effect of different temperatures on the degradation of chloramphenicol by strain CS1:

[0056] In 50 mL of inorganic salt medium with a chloramphenicol concentration of 20 mg / L, 21 mg of the degrading agent of strain CS1 was inoculated and cultured at 150 rpm for 90 minutes at 5, 15, 25, 30, 35, 40, 50, 60 °C respectively. As Figure 8 shown, at 30 °C, chloramphenicol was completely removed in 60 minutes; at 35 and 40 °C, chloramphenicol was completely removed in 75 minutes; at 50 °C, chloramphenicol was completely removed in 90 minutes. At 15 - 50 °C, strain CS1 maintained high-efficiency chloramphenicol degradation activity, and the degradation rate of chloramphenicol was the highest at 30 °C.

[0057] Example 5

[0058] Effect of different pH values on the degradation of chloramphenicol by strain CS1:

[0059] In 50 mL of inorganic salt medium (chloramphenicol concentration is 20 mg / L) with pH values of 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0 respectively, 10 mg of the degrading agent of strain CS1 was inoculated and cultured at 30 °C and 150 rpm for 90 minutes. As Figure 9 shown, the degradation rates of chloramphenicol after 90 minutes were 21.2%, 46.6%, 52.5%, 85.5%, 97.5%, 98.6%, 78.9%, 72.4%, 47.8% respectively. The optimal pH value for strain CS1 to degrade chloramphenicol is 7.

[0060] Example 6

[0061] Determination of the mineralization effect of chloramphenicol in paddy soil cultured at 15 °C by strain CS1:

[0062] Paddy soil samples were collected from Changshu, Jiangsu, air-dried, ground and sieved (<0.85 mm). The soil organic matter content was 33 g / kg, total nitrogen was 2.2 g / kg, cation exchange capacity was 23.4 cmol / kg, and the pH value was 7.30. The soil moisture content was adjusted to 40%, and 14C-labeled chloramphenicol (Moravek Inc., USA) was added to make the chloramphenicol concentration in the soil 5 mg / kg (60000 Bq / kg). The treatment group was additionally added with the degrading agent of strain CS1 and mixed evenly to make the CS1 concentration in the soil 1×109 CFU / g soil, placed in an incubator at 15 °C and incubated under dark conditions at a constant temperature. The amount of 14CO2 produced was monitored daily using a liquid scintillation counter for the first 6 days. Each group was replicated 3 times. The mineralization rate reached 64.8% after 6 days; after 30 days, 5 mg / kg (72000 Bq / kg) of chloramphenicol was added, and the monitoring continued for 6 days. The mineralization rate reached 66.5% after 6 days ( Figure 10 ).

[0063] Example 7

[0064] Determination of the mineralization effect of strain CS1 on chloramphenicol in red soil incubated at 30 °C:

[0065] Red soil samples were collected from Yingtan, Jiangxi, air-dried, ground and sieved (<0.85 mm). The soil organic matter content was 15 g / kg, total nitrogen was 0.6 g / kg, cation exchange capacity was 5.2 cmol / kg, and the pH value was 4.43. The soil moisture content was adjusted to 30%, and 14C-labeled chloramphenicol (Moravek Inc., USA) was added to make the chloramphenicol concentration in the soil 5 mg / kg (60000 Bq / kg). The treatment group was additionally mixed with a degrading agent of strain CS1 to make the CS1 concentration in the soil 1×10 9 CFU / g soil, placed in an incubator at 30 °C and incubated under dark conditions at a constant temperature. The amount of 14CO2 produced was monitored daily using a liquid scintillation counter for the first 6 days. Each group was replicated 3 times. The mineralization rate reached 68.0% after 6 days; after 30 days, 5 mg / kg (72000 Bq / kg) of chloramphenicol was added, and the monitoring continued for 6 days. The mineralization rate reached 68.6% after 6 days ( Figure 11 )。

[0066] Example 8

[0067] Analysis of the degradation performance of strain CS1 on common chloramphenicol and p-nitrobenzene organic pollutants:

[0068] Using thiamphenicol, p-nitrocinnamic acid and p-nitrobenzoic acid ester at 20 mg / L in an inorganic salt medium as the sole carbon source respectively, the degradation ability of strain CS1 on these common organic pollutants was investigated. The experimental results showed that strain CS1 could degrade the above-mentioned organic pollutants to varying degrees, and could degrade 100% of thiamphenicol, p-nitrobenzoic acid ester and 71% of p-nitrocinnamic acid within 10 hours ( Figure 12 )。

[0069] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the present invention, and all of them should be covered within the scope of the present invention.

Claims

1. Nocardia testacea CS1 was deposited at the China Center for Type Culture Collection on March 10, 2025, with the deposit number CCTCC M 2025425 and the deposit address at Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

2. A degrading bacterium agent, characterized in that it contains the Nocardia testacea described in claim 1.

3. Application of the Nocardia testacea described in claim 1 or the bacterium agent described in claim 2 in repairing the environment polluted by chloramphenicol, thiamphenicol and nitrobenzene compounds.

4. The application according to claim 3, wherein: The Nocardia testacea described above or the bacterium agent described in claim 2 is added to an environment containing chloramphenicol, thiamphenicol and nitrobenzene compounds for reaction.

5. The application according to claim 4, wherein: The reaction temperature is 15 - 50 °C.

6. The application according to claim 4, wherein: The reaction pH is 4 - 11.

7. The application according to claim 5, wherein: The reaction temperature is 30 °C.

8. The application according to claim 6, characterized in that: The reaction pH is 7 - 8.

9. The application according to claim 3, wherein: The concentration of chloramphenicol in the environment is 100 mg / L.

10. Application of the Nocardia testacea described in claim 1 in preparing products for removing chloramphenicol, thiamphenicol and nitrobenzene compounds from water and soil.

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